Monoamine oxidase-A (MAO-A) is an enzyme that has historically been linked to major depressive disorder (MDD). The prevalence of MDD among breast cancer patients is almost 25%, but realistically it is underdiagnosed within this patient population. Most breast cancer is deemed estrogen receptor positive [ER(+)] and is commonly treated with the anti-estrogenic chemotherapeutic compound tamoxifen. Resistance to tamoxifen has been associated with a paradoxical activation of the stress-associated kinase, p38(MAPK) (normally associated with cell death). Our research group has recently demonstrated that p38(MAPK) can regulate the function of MAO-A in glial cells. Taken together, MAO-A, depression and p38(MAPK) are all associated with a poor prognosis in breast cancer patients, particularly those with an ER(+) status. Several mechanisms have been proposed in each respect and we hope to further elucidate this relationship by focussing on the interaction between MAO-A and p38(MAPK) in the context of breast cancer. The hypothesis states that a functional interaction between the p38(MAPK) and MAO-A systems alters breast cancer cells in an ER-dependent manner. The proposed objectives of this project are to determine what might be influencing MAO-A function in breast cancer cells, and how changes in MAO-A function affect cell phenotype. Using pharmacological approaches (i.e. antidepressant drugs), we investigated the role of MAO-A and p38(MAPK) on selected characteristics of ER(+) (e.g. MCF-7) and ER(-) (e.g. MDA-MB-231) breast cancer cells under four treatment conditions, which include clorgyline (CLG), an antidepressant MAO-A inhibitor, and SB203580, an inhibitor of p38(MAPK). Our results indicate that the very high MAO-A activity in MDA-MB-231 (MB-231) cells was partly dependent on p38(MAPK) activity. The tumourigenic properties (e.g. anchorage-independent growth, migration) of MB-231 cells depended on both MAO-A and p38(MAPK) functions, although the effects were not additive suggesting that both inhibitors were exerting their respective effects via common signalling targets. The role of MAO-A and p38(MAPK) on MB-231 mitochondrial function and cell growth was negligible. In contrast, MAO-A and p38(MAPK) only influenced mitochondrial function in MCF-7 cells and did not affect any of the other tumourigenic properties measured. Immunocytochemical methods, supported by Western blotting, revealed an increase in E-cadherin expression in both cell lines. This suggested that MAO-A and p38(MAPK) could be influencing transitions between epithelial and mesenchymal phenotypes. Our in vitro findings suggest that MAO-A and p38(MAPK) might contribute to a common mechanism in breast cancer cell lines, but that their influence on cell phenotype is less dependent on the respective cell's ER status and perhaps more so dependent on the cell's metastatic potential. If this is the case, then the contribution of MAO-A and p38(MAPK) to [clinical] metastatic breast cancer should be duly considered. Our ongoing investigations are focussing on the influence of clinically relevant antidepressants on breast cancer cell phenotype in vitro.

This project focused on determining the effect of lithium on glucose uptake, glycogen synthesis, and insulin signaling proteins, protein kinase B (Akt1) and GSK-3, in isolated soleus muscle from female lean Zucker rats. We also investigated the role of the stress-activated p38 MAPK in the action of lithium to activate skeletal muscle glucose transport. In the absence of insulin, lithium (10 mM LiCl) increased basal glucose transport by 62% (p<0.05) and glycogen synthesis by 112%. Lithium did not alter phosphorylation of Akt ser473, but enhanced GSK-3β ser9 phosphorylation by 41%. Lithium further enhanced the effect of insulin on glucose transport (42%), glycogen synthesis (44%), and GSK-3ß phosphorylation (13%). Lithium increased phosphorylated p38 MAPK 31% without and 19% with insulin. Moreover, a selective p38 MAPK inhibitor, A304000, completely prevented the lithium-induced enhancement of glucose transport revealing the critical involvement of p38 MAPK phosphorylation in lithium-induced glucose transport in isolated skeletal muscle.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic, inflammatory condition, characterised by airflow limitation. The use of glucocorticoids (GC) as an anti-inflammatory treatment in COPD has limited clinical benefits, and as such, new treatments are needed. Identifying key pathways involved in the inflammatory response in COPD may enable the development of novel treatments. The aims of this thesis were to examine the steroid sensitivity of an in vitro mixed sputum culture cell model, comparing COPD cells to smoking and non-smoking controls, examine expression of the intracellular signalling molecule p38 Mitogen Activated Protein Kinase (MAPK) in COPD lungs compared with controls, examine the GC and p38 MAPK inhibitor and dual therapy sensitivity of a bronchial epithelial cell line and finally, to understand the mechanisms by which a p38 MAPK inhibitor in combination with a GC synergistically inhibit pro-inflammatory mediator production in a bronchial epithelial cell line. Dexamethasone inhibits mixed sputum cell pro-inflammatory mediator release, with no differences in sensitivity observed between COPD and control cells. Isolated sputum neutrophils demonstrate modest sensitivity to dexamethasone, which is in contrast to blood neutrophils. There are increased numbers of cells positive for activated p38 MAPK in COPD lungs compared with controls, specifically localised to follicular B and CD8+ T cells, bronchial epithelial cells and alveolar and sputum macrophages. Lung and sputum neutrophils are devoid of activated p38 MAPK, and a pharmacological p38 MAPK inhibitor has no effect on pro-inflammatory mediator production from these cells. This is in contrast to blood neutrophils, whereby p38 MAPK activation can be induced following LPS stimulation and in vitro cell culture, and pro-inflammatory mediator release is inhibited by a p38 MAPK inhibitor. Dexamethasone and birb 796 inhibit stimulated pro-inflammatory mediator release from a bronchial epithelial cell line in a dose-dependent manner. Sensitivity to either drug is dependent on stimuli and the pro-inflammatory mediator analysed. There is additive and synergistic inhibition of pro-inflammatory mediator production when combination therapy comprising dexamethasone and birb 796 is used compared with either drug alone. This may be due to Birb 796 enhancing dexamethasone-mediated nuclear translocation of the glucocorticoid receptor, which may enhance the GC-mediated anti-inflammatory effects. Combination therapy may therefore be a useful therapeutic in the treatment of COPD.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25498号 / 医博第5098号 / 新制||医||1074(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 寺田 智祐, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Células dendríticas são as principais células apresentadoras de antígeno e apresentam alterações em pacientes com câncer. As vias de sinalização ERK 1/2 e p38 MAPK participam da diferenciação de DCs derivadas de monócitos (Mo-DCs). A exposição ao sobrenadante tumoral (ST) da linhagem MCF-7 levou à diminuição de CD1a e aumento de CD14 (frequência), além do aumento de IL-6 e IL-10. A inibição da via ERK1/2 MAPK corrigiu a expressão de CD14 e corrigiu parcialmente a produção das citocinas. A inibição da via p38 MAPK corrigiu a expressão de CD1a e CD14 e diminuiu parcialmente a produção das citocinas. Identificamos a proteína de choque térmico HSP27. A exposição à HSP27 não levou às alterações observados quando as células foram expostas ao ST. Por fim, em Mo-DCs de pacientes com câncer de mama o tratamento com o inibidor da p38 MAPK diminuiu a expressão de CD86 e HLA-DR. Portanto, os resultados deste trabalho sugerem que a inibição da via p38 MAPK não parece ser uma abordagem interessante na manipulação de Mo-DCs de pacientes com carcinoma ductal invasivo de mama. / Dendritic cells are the main presenting cells and present alterations in cancer patients. The signaling pathways p38 and ERK1/2 MAPK participate of monocyte-derived dendritic cells (Mo-DCs) differentiation. Exposition to MCF-7s supernatant (TS) decreased CD14 and CD1a expression (frequency) while enhanced IL-6 and IL-10 production. Inhibition of ERK1/2 MAPK reverted CD14 expression and partially reverted cytokines production. Inhibition of p38 MAPK reverted CD1a and CD14 expression and partially reverted cytokines production too. We identified the heat shock protein HSP27. Exposition to HSP27 did not cause the observed alterations seen when the cells were exposed to TS. Lastly, treatment of Mo-DCs from breast cancer patients with the p38 inhibitor decreased CD86 and HLA-DR expression. Therefore, the data presented in this study suggest that p38 MAPK inhibition does not appear to be an interesting approach in the manipulation of Mo-DCs from breast cancer patients.

Células dendríticas são as principais células apresentadoras de antígeno e apresentam alterações em pacientes com câncer. As vias de sinalização ERK 1/2 e p38 MAPK participam da diferenciação de DCs derivadas de monócitos (Mo-DCs). A exposição ao sobrenadante tumoral (ST) da linhagem MCF-7 levou à diminuição de CD1a e aumento de CD14 (frequência), além do aumento de IL-6 e IL-10. A inibição da via ERK1/2 MAPK corrigiu a expressão de CD14 e corrigiu parcialmente a produção das citocinas. A inibição da via p38 MAPK corrigiu a expressão de CD1a e CD14 e diminuiu parcialmente a produção das citocinas. Identificamos a proteína de choque térmico HSP27. A exposição à HSP27 não levou às alterações observados quando as células foram expostas ao ST. Por fim, em Mo-DCs de pacientes com câncer de mama o tratamento com o inibidor da p38 MAPK diminuiu a expressão de CD86 e HLA-DR. Portanto, os resultados deste trabalho sugerem que a inibição da via p38 MAPK não parece ser uma abordagem interessante na manipulação de Mo-DCs de pacientes com carcinoma ductal invasivo de mama. / Dendritic cells are the main presenting cells and present alterations in cancer patients. The signaling pathways p38 and ERK1/2 MAPK participate of monocyte-derived dendritic cells (Mo-DCs) differentiation. Exposition to MCF-7s supernatant (TS) decreased CD14 and CD1a expression (frequency) while enhanced IL-6 and IL-10 production. Inhibition of ERK1/2 MAPK reverted CD14 expression and partially reverted cytokines production. Inhibition of p38 MAPK reverted CD1a and CD14 expression and partially reverted cytokines production too. We identified the heat shock protein HSP27. Exposition to HSP27 did not cause the observed alterations seen when the cells were exposed to TS. Lastly, treatment of Mo-DCs from breast cancer patients with the p38 inhibitor decreased CD86 and HLA-DR expression. Therefore, the data presented in this study suggest that p38 MAPK inhibition does not appear to be an interesting approach in the manipulation of Mo-DCs from breast cancer patients.

<p>Multiple signaling pathways control protein synthesis by modulating translation initiation factors. Map Kinase Integrating Kinase 1 (Mnk1) relays signals to its major downstream target eIF4E. Activation of Mnk1 and subsequent phosphorylation of eIF4E results in changes in translation rates for subsets of mRNAs. Both the Erk1/2 and p38 MAPK pathways activate Mnk1 meaning that Mnk1 responds to growth signals through Erk1/2 and stress signals through p38 MAPK. However, it is not clear how Mnk1 mediates translational changes specific to each pathway. We investigated the activation of Mnk1 by stress and cytokines through the p38 MAPK pathway. We found that of the four different p38 MAPK isoforms, p38&alpha; alone controls acute stress and cytokine signaling to translation machinery. Furthermore, this regulatory axis is greatly diminished in neurons. We discovered that p38&alpha; expression is repressed in the brain due to two neuron-selective microRNAs, miR-124 and -128. Next, we investigated the mechanism of p38&alpha; mediated Mnk1 activation to see if it differed from Erk1/2 mediated activation. Looking at the induced binding of Mnk1 to eIF4G, we found that the dissociation rate varies depending on the activating pathways. This shows that Mnk1 is not a true convergence point of p38 and Erk1/2 MAPK pathways resulting in identical downstream effects, but that Mnk1 mediates pathway specific effects on translation factors.</p> / Dissertation

The small GTPases function as molecular switches to control diverse signaling cascades. The mammalian Rit and Rin, along with Drosophila Ric, comprise an evolutionarily conserved subfamily of the Ras-related GTPases. Previous studies using cultured cell models suggested that Rit was involved in the control of cell proliferation, transformation, neuronal differentiation, morphogenesis, and cell survival, but the principal physiological function of Rit remained uncharacterized. To address this outstanding question, we employed a genetic approach, engineering a Rit knockout mouse. Using this animal model, we demonstrate a central role of Rit in governing cell survival in a p38-dependent fashion. Primary mouse embryonic fibroblasts (MEFs) derived from Rit-/- mice display increased apoptosis and selective disruption of MAPK signaling following oxidative stress. These deficits include a reduction in ROS-mediated stimulation of a novel p38-MK2-HSP27 signaling cascade, which appears to act upstream of the mTORC2 complex to control Akt-dependent cell survival. In the adult brain, proliferation of stem cells within the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), provide a lifelong supply of new neurons. Adult neurogenesis appears critical for learning and memory and is altered in animal models of brain injury and neurological diseases. Thus, a greater understanding of the regulation of adult neurogenesis will provide insight into its myriad physiological roles but also to the development of therapeutic strategies for the treatment of injury and the progression of brain diseases. Here we find that Rit plays a central role in governing the survival of hippocampal neurons in response to oxidative stress. Importantly, using a controlled cortical impact model of traumatic brain injury (TBI), we show that Rit acts to protect newborn immature neurons within the SGZ of the DG from apoptosis following TBI. Finally, studies indicate that Rit plays a significant role in directing IGF-1 signaling, a key neurotrophin known to promote neurogenesis and to protect neurons against apoptotic stress. Together, these studies establish Rit as a critical regulator of a p38 MAPKdependent signaling cascade that functions as an important survival mechanism for cells in response to oxidative stress, including the survival of newborn hippocampal neurons in the traumatically injured brain.

Pain from bone metastases is multifaceted with clinical descriptors including ongoing pain, hypersensitivity to external stimuli and intermittent episodes of breakthrough pain characterized as a sudden and abrupt onset of severe pain on a background of well-controlled pain. Moreover, cancer-induced bone pain remains inadequately managed due to a myriad of side effects associated with the current pain relieving regimens, which primarily rely on administration of opiates. Despite advances made in cancer therapeutics, these patients experience an inferior quality of life with incapacitating pain with limited daily activities. Development of long-term novel, non-opiate mechanism-based therapeutics with limited side effects is considered beneficial in elevating the patients' quality of life. First part of this dissertation encompasses the role of p38 MAPK in a mouse model of cancer-induced bone pain in which breast cancer cells were injected and sealed into the femur. Our data demonstrated that both acute and prolonged inhibition of p38 MAPK blocked cancer-induced spontaneous pain but had no effect on the evoked pain indicating important differences in mechanisms mediating ongoing pain as opposed to evoked pain. Undermanaged control of breakthrough pain is attributed to poor understanding of underlying mechanisms and how they may differ from ongoing pain due, in part, to lack of a pre-clinical model in which these mechanisms can be studied. We have established a rat model of cancer-induced bone pain to examine ongoing pain and pain relief using conditioned place preference paradigm as well as breakthrough pain using palpation-induced conditioned place aversion. We have shown that while peripheral afferent input from the tumor-bearing tibia mediates cancer-induced ongoing pain and initiation of breakthrough pain, it does not contribute to the maintenance of breakthrough pain. These data suggest that molecular targets mediating these two mechanisms may be different. This hypothesis was confirmed by our findings in this model that acute blockade of interleukin-6 blocked movement-evoked breakthrough pain in tumor-bearing rats, but failed to block tumor-induced ongoing pain. Hence, we provide a platform to manipulate treatments that can be given alone or in combination with opiates in such a way that patients receive adequate control of breakthrough pain.

Many pathways dysregulated in prostate cancer are also involved in epithelial differentiation. To better understand prostate tumor initiation, we sought to investigate specific genes and mechanisms required for normal basal to luminal cell differentiation. Utilizing human prostate basal epithelial cells and an in vitro differentiation model, we tested the hypothesis that regulation of NOTCH3 by the p38 MAPK family (hereafter p38-MAPK), via MYC, is required for luminal differentiation. Inhibition (SB202190 and BIRB796) or knockdown of p38a (also known as MAPK14) and/or p38d (also known as MAPK13) prevented proper differentiation. Additionally, treatment with a gamma-secretase inhibitor (RO4929097) or knockdown of NOTCH1 and/or NOTCH3 greatly impaired differentiation and caused luminal cell death. Constitutive p38-MAPK activation through MKK6(CA) increased NOTCH3 (but not NOTCH1) mRNA and protein levels, which was diminished upon MYC inhibition (10058-F4 and JQ1) or knockdown. Furthermore, we validated two NOTCH3 enhancer elements through a combination of enhancer (e) RNA detection (BruUV-seq) and luciferase reporter assays. Finally, we found that the NOTCH3 mRNA half-life increased during differentiation or upon acute p38-MAPK activation. These results reveal a new connection between p38-MAPK, MYC and NOTCH signaling, demonstrate two mechanisms of NOTCH3 regulation and provide evidence for NOTCH3 involvement in prostate luminal cell differentiation.